Delivery of endolysin across outer membrane of Gram-negative bacteria using translocation domain of botulinum neurotoxin

The emergence of multidrug-resistant pathogens has outpaced the development of new antibiotics, leading to renewed interest in endolysins. Endolysins have been investigated as novel biocontrol agents for Gram-positive bacteria. However, their efficacy against Gram-negative species is limited by the barrier presented by their outer membrane, which prevents endolysin access to the peptidoglycan substrate. Here, we used the translocation domain of botulinum neurotoxin to deliver endolysin across the outer membrane of Gram-negative bacteria. The translocation domain selectively interacts with and penetrates membranes composed of anionic lipids, which have been used in nature to deliver various proteins into animal cells. In addition to the botulinum neurotoxin translocation domain, we have fused bacteriophage-derived receptor binding protein to endolysins. This allows the attached protein to efficiently bind to a broad spectrum of Gram-negative bacteria. By attaching these target-binding and translocation machineries to endolysins, we aimed to develop an engineered endolysin with broad-spectrum targeting and enhanced antibacterial activity against Gram-negative species. To validate our strategy, we designed engineered endolysins using two well-known endolysins, T5 and LysPA26, and tested them against 23 strains from six species of Gram-negative bacteria, confirming that our machinery can act broadly. In particular, we observed a 2.32 log reduction in 30 min with only 0.5 µM against an Acinetobacter baumannii isolate. We also used the SpyTag/SpyCatcher system to easily attach target-binding proteins, thereby improving its target-binding ability. Overall, our newly developed endolysin engineering strategy may be a promising approach to control multidrug-resistant Gram-negative bacterial strains.